(1) The physical and catalytic properties of a novel magnesium-dependent, calcium-inhibited protein phosphatase has been further characterized. Although 3 bands of 39,000, 25,000, and 16,000 molecular weight, were observed on sodium dodecyl sulfate gel electrophoresis, the two smaller bands were shown to be proteolyzed fragments of the larger band by experiments involving limited proteolysis and immunoblotting. The phosphatase was also shown not to attack phosphoseryl residues on either alpha or beta subunit of phosphorylase kinase and not inhibited by phosphatase inhibitor 2 or okadaic acid, thereby demonstrating that it is not a type 1 or type 2 phosphatase. Other properties of the phosphatase studied are: extinction coefficient at 280nm, pH optimum, inhibition constants for calcium in the presence and absence of a protein activator, effect of various divalent cations, several brain phosphoprotein as potential substrates, and further support for the enzyme being a calcium binding protein. A 120,000 molecular weight protein activator is found to be capable of 30 to 100-fold activation of the calcium-inhibited phosphatase. (2) A method for analyzing kinetic parameters for two interacting components present at comparable levels has been developed and applied to the study of B1-B2 interaction and inhibition by various synthetic peptides in the Escherichia coli ribonucleotide reductase system. (3) A method for differentiating inter- or intramolecular autophosphorylation and autodephosphorylation has been proposed. The method is based on the change of apparent first-order rate constant obtained at different kinase or phosphatase concentrations.